Design and facile production of recombinant resilin-like polypeptides: gene construction and a rapid protein purification method

Abstract

Resilin is an elastic protein found in specialized regions of the cuticle of insects, which displays unique resilience and fatigue lifetime properties. As is the case with many elastomeric proteins, including elastin, gliadin and spider silks, resilin contains distinct repetitive domains that appear to confer elastic properties to the protein. Recent work within our laboratory has demonstrated that cloning and expression of exon 1 of the Drosophila melanogaster CG15920 gene, encoding a putative resilin-like protein, results in a recombinant protein that can be photochemically crosslinked to form a highly resilient, elastic biomaterial (Rec1 resilin). The current study describes a recursive cloning strategy for generating synthetic genes encoding multiple copies of consensus polypeptides, based on the repetitive domains within resilin-like genes from D. melanogaster and Anopheles gambiae. A simple non-chromatographic purification method that can be applied to these synthetic proteins and Rec1 is also reported. These methods for the design and purification of resilin-like periodic polypeptides will facilitate the future investigation of structural and functional properties of resilin, and the development of novel highly resilient biomaterials.

Molecular and functional characterisation of resilin across three insect orders

“…A "heat and salting out" method was applied to clarified soluble fractions of cell lysates as previously published Lyons et al, 2007). Both recombinant proteins were heat stable at 80 C for 10 min, and precipitated at 20% ammonium sulphate.…”

“…For these reasons, we have decided initially to express the B isoforms. Both were expressed as soluble proteins which were amenable to the "heat and salting out" purification method as previously used for Rec1 resilin and another intrinsically-unstructured protein An16 Lyons et al, 2007). Resulting recombinant proteins were pure as determined by SDS-PAGE analysis and sequence analysis by mass spectroscopy.…”

Molecular and Supramolecular Structural Studies on Significant Repetitive Sequences of Resilin

“…[21] Furthermore, high yield recombinant synthesis of resilin-like polypeptide has been reported, containing multiple copies of a repeat sequence within the resilin-like gene of D. melanogaster and Anopheles gambiae (African malaria mosquito). [22,23] The presence of repeated sequences in common with many other elastomeric proteins, prompted us to investigate the molecular structure of this protein by the use of model polypeptides based upon two different D. melanogaster resilin-repeat sequences. This approach has been successfully applied to elastin, abductin, lamprin, flagelliform silk and other elastomeric proteins, and used to identify multiconformational equilibria among poly-l-proline II (PPII), b-strands and b-turn conformations.…”

Self-organization, interfacial interaction and photophysical properties of gold nanoparticle complexes derived from resilin-mimetic fluorescent protein rec1-resilin

“…The final properties of the protein-AuNP hybrids are very sensitive not only to the chemical composition of the protein and the size of AuNPs; but also on their topological organization including the distance of the fluorophores from AuNP surfaces. Herein, we report the synthesis of AuNPs/rec1-resilin bioconjugates of size specificity, aggregation and unique photophysical properties using pre-organized engineered biomimetic protein rec1-resilin [24] as a nanoreactor.…”

“…The elasticity of resilin is best known for its roles in insect flight and the remarkable jumping ability of fleas, and spittle bugs. Recently, we have reported the synthesis of a resilinmimetic polypeptide rec1-resilin from the repeat sequences of the first exon of the Drosophila melanogaster CG15920 gene using recombinant DNA technology [24,25]. Its physical, physiochemical and thin film characteristics have also been discussed in detail [27e29].…”